Lysimachia Fanii, a New Species of Primulaceae from Limestone Area of Guangxi, China

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A peer-reviewed open-access journal PhytoKeys 130: 75–84 (2019) Lysimachia fanii, a new species from China 75 doi: 10.3897/phytokeys.130.34655 RESEARCH ARTICLE http://phytokeys.pensoft.net Launched to accelerate biodiversity research

Lysimachia fanii, a new species of Primulaceae from limestone area of Guangxi, China

Yun-Feng Huang1, Li-Na Dong2, Wei-Bin Xu2

1 Guangxi Key Laboratory of Traditional Chinese Medicine Quality Standards, Guangxi Institute of Chinese Medicine & Pharmaceutical Sciences, Nanning 530022, Guangxi, China 2 Guangxi Key Laboratory of Plant Conservation and Restoration Ecology in Karst Terrain, Guangxi Institute of Botany, Guangxi Zhuang Auto- nomous Region and Chinese Academy of Sciences, Guilin 541006, Guangxi, China

Corresponding author: Li-Na Dong ([email protected]); Wei-Bin Xu ([email protected])

Academic editor: Wen-Bin Yu | Received 19 March 2019 | Accepted 19 May 2019 | Published 29 August 2019

Citation: Huang Y-F, Dong L-N, Xu W-B (2019) Lysimachia fanii, a new species of Primulaceae from limestone area of Guangxi, China. In: Cai J, Yu W-B, Zhang T, Li D-Z (Eds) Revealing of the plant diversity in China’s biodiversity hotspots. PhytoKeys 130: 75–84. https://doi.org/10.3897/phytokeys.130.34655

Abstract Lysimachia fanii, a new species of Lysimachia (Subgen. Idiophyton, Primulaceae), is described and illus- trated from Guangxi, China based on morphological and molecular data. Lysimachia fanii differs from L. verbascifolia, L. rupestris and L. alpestris mainly by the habit being nearly rosulate, leaves congested at the apex of the rhizome, leaf blades spatulate to narrowly oblanceolate and flowers solitary. Phylogenetic analyses supported L. verbascifolia as sister to L. fanii. This new species is endemic to limestone areas in Liucheng county of Guangxi, China.

Keywords Lysimachia, subgen. Idiophyton, Primulaceae, taxonomy, limestone flora

Introduction

The genusLysimachia L. (1753: 146) includes about 190 species and was originally placed in Primulaceae (Cronquist 1981, Takhtajan 1997), but later transferred into Myrsinaceae, based on morphological and molecular evidence (Anderberg and Ståhl 1995, Anderberg et al. 1998, 2002, Källersjö et al. 2000, Hao et al. 2004). Mysi- naceae was later merged into Primulaceae s.l., hence Lysimachia was replaced into

Copyright Yun-Feng Huang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 76 Yun-Feng Huang et al. / PhytoKeys 130: 75–84 (2019)

Primulaceae (China Phylogeny Consortium 2016). The majority of species within the genus are distributed in temperate and subtropical regions of the Northern Hemi- sphere, with some species in Africa, Australia and South America. In China, the genus has 138 species (Hu and Kelso 1996) and is highly diversified in south-western China, especially in limestone areas. According to the flower and gland morphology, the genus is separated into five subgenera, viz. subgen. Idiophyton Hand.-Mazz., subgen. Lysimachia, subgen. Palladia (Moench) Hand.-Mazz., subgen. Heterostylan- dra (Hand.-Mazz.) F.H.Chen & C.M.Hu and subgen. Naumburgia (Moench) Klatt. (Chen and Hu 1979, Chen et al. 1989). The south-western limestone karst area is one of China’s biodiversity hotspots. These areas are fragile and sensitive to environmental change and, in the wake of the rapid economic development of China, they are facing serious threat. Documenta- tion of the plant diversity in these regions is urgently needed. Thus, we are surveying traditional medicinal plants in the limestone areas of Guangxi and trying to increase our knowledge of these poorly studied areas. During fieldwork in May 2018, we discovered an unknown species in Lysimachia. This species is allied to subgen. Het- erostylandra by having rosette leaves, but it differs in having heteromorphic flowers. It shows alliance to subgen. Idiophyton, subgen. Lysimachia and subgen. Palladia by having 5-merous flowers, but has unique filaments, anthers and glands. After morphological observation and consulting relevant literature (Chen and Hu 1979, Chen et al. 1989, Hu and Kelso 1996, Tong et al. 2017), we confirm that the rare plant is a new species and has been placed into subgen. Idiophyton, based on morphology and molecular analyses.

Material and methods Taxon sampling

We followed the classification of Lysimachia of Chen et al. (1989) and Hu and Kel- so (1996). Leaves were collected from the holotype (L.Y. Fan et al., FLY2018001 in GXMI) and paratypes (L.Y. Fan et al., FLY2018002 in IBK & GXMI) to represent the new species. Twenty related taxa within subgen. Idiophyton, one taxon within subgen. Heterostylandra and four taxa within subgen. Lysimachia were selected to ascertain the phylogenetic relationships within Lysimachia (Anderberg et al. 2002). Based on Yan et al. (2018), Pelletiera verna A. St.-Hil. and Anagallis monelli L. were selected as outgroups.

DNA sequencing

Total genomic DNA was extracted from silica-dried plant leaves by a modified CTAB protocol (Doyle and Doyle 1987). Four chloroplast DNA regions (atpF-atpH, rpl32- trnL, trnL-F and trnS-trnG) and one nuclear loci (ITS) were selected and amplified following Yan et al. (2018). Genebank Accession Numbers are listed in Table 1. Lysimachia fanii, a new species from China 77

Table 1. Species of Lysimachia and related taxa sampled and GenBank accession numbers of sequences used in this study.

Taxa atpF-atpH rpl32-trnL trnL-F trnS-trnG ITS Anagallis monelli MG950735 MG950945 MG951268 MG951373 MG877752 L. alpestris MG950743 MG950953 MG951276 MG951381 MG877760 L. baviensis MG950773 MG950983 MG951306 MG951410 MG877790 L. capillipes MG950748 MG950958 MG951281 MG951386 MG877765 L. chapaensis MG950749 MG950959 MG951282 MG951387 MG877766 L. confertifolia MG950757 MG950967 MG951290 – MG877774 L. crispidens MG950759 MG950969 MG951292 MG951396 MG877776 L. engleri MG950765 MG950975 MG951298 MG951402 MG877782 L. foenum-graecum MG950770 MG950980 MG951303 MG951407 MG877787 L. heterobotrys MG950779 MG950989 MG951311 MG951415 MG877796 L. insignis MG950784 MG950994 MG951316 MG951420 MG877801 L. lancifolia MG950788 MG960998 MG951320 MG951424 MG877805 L. laxa MG950789 MG950999 MG951321 MG951425 MG877806 L. longipes MG950792 MG951002 MG951324 MG951428 MG877809 L. microcarpa MG950796 MG951006 MG951328 MG951432 MG877813 L. millietii MG950797 MG951007 MG951329 MG951433 MG877814 L. nemorum MG950799 MG951009 MG951331 MG951435 MG877816 L. nutantiflora MG950801 MG951011 MG951333 MG951437 MG877818 L. peduncularis MG950805 MG951015 MG951337 – MG877822 L. petelotii MG950808 MG951018 MG951340 – MG877825 L. pittosporoides MG950810 MG951020 MG951342 MG951445 MG877827 L. punctata MG950813 MG951023 MG951345 MG951448 MG877830 L. trichopoda MG950826 MG951038 MG951359 MG951461 MG877845 L. verbascifolia MG950827 MG951039 MG951360 MG951462 MG877846 L. vittiformis MG950828 MG951040 MG951361 MG951463 MG877847 L. vulgaris MG950829 MG951041 MG951362 MG951464 MG877848 Pelletiera verna MG950832 MG951044 MG951365 MG951467 MG877851 L. fanii 01 MK516268 MK516270 MK516272 – MK516275 L. fanii 02 MK516269 MK516271 MK516273 MK516274 MK516276

Phylogenetic analysis

Sequences of each DNA region were aligned using MUSCLE 3.8.31 (Edgar 2004a, 2004b) and adjusted manually where necessary. Indels were treated as gaps and all regions were combined as a single region for further study. Maximum Parsimony (MP) analyses were conducted using PAUP v.4.0b10 (Swof- ford 2002). Heuristic searches were carried out with 1000 replicates and tree-bisection- reconnection (TBR) branch swapping. A strict consensus tree was summarised from all the most parsimonious trees. Node support was assessed by 500 bootstrap replicates using TBR branch swapping. Bayesian Inference (BI) analyses were conducted using MrBayes version 3.1.2 (Ron- quist and Huelsenbeck 2003). The Markov chain Monte Carlo (MCMC) chains were run for 100 000 generations while trees were sampled every 100 generations. The MCMC chains were stopped when the average standard deviation of the split frequencies was 0.008 after 100 000 generations, which meant that the chains were converged to a stationary distribution. A majority-rule consensus tree was constructed after removing a burn-in of 25% of the trees. Posterior Probability (PP) values were used to estimate branch support. 78 Yun-Feng Huang et al. / PhytoKeys 130: 75–84 (2019)

Figure 1. Phylogenetic tree inferred by MP and BI analyses based on the combined dataset of four plastid loci (atpF-atpH, rpl32-trnL, trnL-F and trnS-trnG) and nuclear ITS. Numbers above branches indicate maximum parsimony bootstrap/Bayesian inference posterior probability.

Results Molecular systematic relationship

In total, 29 atpF-atpH, rpl32-trnL, trnL-trnF and ITS sequences and 25 trnS-G sequences were included. The combined matrix has a length of 3649 aligned characters (ITS: 653bp, atpF-atpH: 512bp, rpl32-trnL: 728bp, trnL-trnF: 946bp, trnS-G: 810bp), of which 363 are parsimony informative. The inferred phylogenies using MP and BI analyses are congruent (Fig. 1). The two samples of the new species (L. fanii) Lysimachia fanii, a new species from China 79 are clustered into subgenus Idiophyton with strong support values in both MP and BI analyses (BS= 100%, PP = 0.99). L. verbascifolia is placed as the sister group to L. fanii with high support in the BI analysis (PP = 0.92).

Taxonomic treatment Lysimachia fanii Y.Feng Huang, W.B.Xu & L.N.Dong, sp. nov. urn:lsid:ipni.org:names:60479343-2 Figs 2, 3

Type. CHINA. Guangxi Zhuangzu Autonomous Region: Liucheng County, Taiping Town, 23°42'50"N, 109°29'20"E, 320 m a.s.l., 21 May 2018, flowering,L.Y. Fan et al. FLY2018001 (holotype, GXMI!; isotypes, IBK!, GXMI!). Diagnosis. Lysimachia fanii differs from congeneric species in subgen.Idiophyton mainly by the habit being nearly rosulate, leaves congested at the apex of the rhizome, leaf blades spatulate to narrowly oblanceolate and flowers being solitary. Description. Herbaceous perennial, glabrous. Rhizome subterete, 6–8 cm long, 4–6 mm in diameter, branched at the apex of the rhizome. Leaves papery, thickly papery to thinly leathery when dry, spirally arranged, congested at the apex of the rhizome, ± forming a rosette, subsessile, spatulate to narrowly oblanceolate, 6–21 × 0.6–2.0 cm, tapering towards the base, apex acute to obtuse, glabrous adaxially, glandular abaxially, veins invisible on both sides. Flowers solitary, axillary. Pedicel 3.0–6.0 cm long, ca. 1 mm in diameter, densely glandular. Calyx lobes lanceolate, 5–6 × ca. 3 mm, 5 (rarely 6), separate to near the base, apex acuminate, glabrous inside, glandular outside. Corolla yellow, deeply parted, tube 0.5–1.0 mm; lobes broadly ovate, 7.0 × 6.0 mm, apex obtuse, glabrous on both sides. Filaments ca. 1.5 mm long, lower 0.5 mm connate into a tube; anthers 3–3.5 mm long, ca. 1 mm in diameter, basifixed, opening by apical pores. Ovary globose, ca. 1 mm in diameter; style 2.8 mm long, slightly shorter than stamens. Capsule globose, 3.5–4 mm in diameter. Phenology. Flowering from May to June. Etymology. The new species is named after Mr. Li-Yong Fan, who first discovered and collected this rare species. Distribution and habitat. Lysimachia fanii is known only from the type locality in Taiping Town, Liucheng County, Guangxi Zhuangzu Autonomous Region, China (Fig. 4). It grows on moist limestone rock surfaces at the entrance to caves. Additional specimens examined. CHINA. Guangxi: Liucheng County, Taiping Town. 320 m a.s.l., 21 May 2018, L.Y. Fan et al. FLY2018002 (IBK, GXMI). Taxonomic notes. Based on the molecular phylogeny, L. fanii belongs to subgenus Idiophyton, that is also supported by the morphological characters of basifixed anthers, short filaments and anthers open by apical pores.L. fanii is morphologically similar to L. verbascifolia C.M.Hu & L.K.Phan that is endemic to limestone areas in Vietnam (Phan and Hu 2011), but can be easily distinguished by its spatulate to nar- 80 Yun-Feng Huang et al. / PhytoKeys 130: 75–84 (2019)

Figure 2. Lysimachia fanii. A Habit B flower, frontal viewC flower, back view (showing six calyx lobes) D corolla opened showing stamens E calyx and pistil F capsule. (Drawn by X.C. Qu from the holotype). rowly oblanceolate leaf blade and glabrous adaxially and glandular abaxially. L. fanii and L. alpestris Champ. ex Benth. resemble each other in having congested leaves and spatulate to narrowly oblanceolate leaf blades and invisible veins and solitary inflo- rescences but L. fanii differs fromL. alpestris by its rhizome which is branched at the Lysimachia fanii, a new species from China 81

Figure 3. Lysimachia fanii. A Habitat B habit C flower, frontal view D flower, back view E flower, lateral view F stamens. 82 Yun-Feng Huang et al. / PhytoKeys 130: 75–84 (2019) apex without stolons from the base, leaf blade glabrous adaxially and glandular abaxially, basifixed anthers which open by apical pores.L. fanii is also similar to L. rupestris F.H.Chen & C.M.Hu from limestone areas distributed in south-western China and northern Vietnam (Tong et al. 2017), but it can be distinguished from the latter by its rhizome which is branched at the apex and without stolons from the base, leaf blade spatulate to narrowly oblanceolate and glabrous adaxially, lateral veins invisible on both sides. A comparison of the main characters of the four species is shown in Table 2.

Table 2. Comparison of characters amongst Lysimachia fanii, L. verbascifolia, L. rupestris and L. alpestris.

Morphological L. fanii L. verbascifolia L. rupestris L. alpestris traits Rhizome 6–8 cm long, branched at 4–10 cm long, geniculate 2–5 cm long, with stolons 1–4 cm long, with stolons the apex at the base from the base from the base Leaf blade spatulate to narrowly elliptic to broadly elliptic, elliptic-oblance-olate, spatulate to narrowly oblanceolate, 6–21 × 7–17 × 3.5–8.0 cm 3–6.5 × 1.2–2.2 cm oblanceolate, 3–6 × 0.6–2.0 cm 0.6–1.5 cm Leaf indumentum glabrous adaxially, greyish villous on both minutely glandular on dense long coarse greyish glandular abaxially sides both sides hairs on both sides Lateral veins invisible on both sides obvious, densely greyish prominent abaxially invisible on both sides villous Inflorescence flowers solitary subumbellate flowers solitary flowers solitary Corolla yellow, deeply parted, tube pale yellow, divided nearly yellow, divided nearly to yellow, deeply parted, tube 0.5–1.0 mm to the base the base 1–1.5 mm Filaments ca. 1.5 mm long, lower 0.5 ca. 3 mm long, connate ca. 1 mm long, connate ca. 3 mm long, lower 1.5 mm connate into a tube basally into a ring basally into a ring mm connate into a tube Anthers 3-3.5 mm long, basifixed, ca. 5 mm long, basifixed, 4–5 mm long, basifixed, ca. 2 mm long, dorsifixed, opening by apical pores opening by apical pores opening by apical pores opening by lateral slits Flower May to June June to October April to May April

Figure 4. The distribution ofLysimachia fanii in Guangxi, China. Lysimachia fanii, a new species from China 83

Acknowledgements

The authors are grateful to Mr. Xincheng Qu (Guangxi Institute of Chinese Medicine & Pharmaceutical Sciences) for the fine line drawings, to Mr. Xiaomao Huang and Mr. Wanlong Wei for their help in the field, to Dr. Mark Hughes (Royal Botanic Garden, Edinburgh) for his helpful comments. This study was supported by Science & Tech- nology Basic Resources Investigation Program of China (Grant no. 2017FY100100) and also in part by The Chinese Medicine Public Health Service Subsidy Special “Na- tional Chinese Medicine Resources Census Project” (CS 2017, No. 6) and the Con- struction of a Joint Laboratory for Quality Research of Traditional Chinese Medicine in Guangxi and Macao (GuiKe AD17195002).

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